The pluripotency and immortality of mouse embryonic stem (ES) cells have made them attractive for basic studies of regenerative medicine, as well as for gaining molecular insight into cellular differentiation at early developmental stages, which are much more difficult to assess by in vivo
approaches. Artificially regulated transgene expression systems are indispensable tools for studying the molecular biology of the differentiation process. Tc-regulated transgene expression systems, known as Tet-off and Tet-on systems, have been widely applied to a variety of biological materials, including mammalian cells (1
). The Tet-off system is based on a Tc-regulatable transactivator (tTA), which induces transcription in the absence of Tc or its analog doxycycline (Dox) through binding to the hCMV*-1 promoter. This promoter is composed of a Tc-responsive element (TRE) followed by a minimal promoter of the human cytomegalovirus (hCMV) immediate early gene. The tTA protein is a fusion protein composed of the TRE-binding domain of Tc repressor protein and the herpes simplex virus VP16 activation domain (3
). Alternatively, the Tet-on system uses a reverse Tc-regulated transactivator (rtTA) which binds TRE and induces transcription of the transgene in the presence of Dox (4
). The Tet-on system seems to be superior to the Tet-off system, in that the former only requires administration of Dox for the induction of expression, but the Tet-off system has other advantages, including the lower level of leaky expression as background and the lower concentration of Tc or Dox required for regulation of expression (5
). Although studies based on these systems have been performed in ES cells, it is difficult to establish independent ES cell lines harboring Tc-regulatable systems. One problem is that ES cells are very sensitive to the toxicity of proteins carrying strong transactivation domains, such as VP16 (S. Masui and H. Niwa, unpublished data). Although this toxicity can be reduced to some extent by using transactivators with iterated minimum transactivation domains (7
), the success rate for generating cell lines stably expressing tTA or rtTA is very low (8
). In addition, randomly integrated transgenes tend to be unstable in the durability and uniformity of their expression due to the positional effect, which depends on the local chromatin structure, especially in ES cells. Moreover, it is laborious and time-consuming to perform repeated electroporations, which use large numbers of cells and large quantities of plasmid DNA, and subsequently evaluate the large number of clones generated for the magnitude and uniformity of induced expression. These findings suggest that a knock-in strategy may be better for ensuring a stable level of transgene expression.
locus was first described as a gene-trapped locus on chromosome 6, from which β-geo was shown to be expressed ubiquitously in the whole body of a mouse (9
). This locus is now regarded as one from which proteins can be expressed ubiquitously at a moderate level. A knock-in strategy into the ROSA26
locus, however, still requires homologous recombination by electroporation and genomic Southern analysis of large number of clones to confirm the identity and location of the inserted gene(s).
The recombinase-mediated cassette exchange (RMCE) system, which uses Cre recombinase and mutant lox
sequences, has become an alternative to homologous recombination as a method for efficiently introducing a transgene into a cassette-acceptor (CA) locus, previously established to carry lox
P and mutant lox
). Among the lox
P mutants, lox
PV (initially designated as lox
2272) has been shown to be suitable for the RMCE reaction, since its recombination efficiency with wild-type lox
P was negligible, but its self-recombination efficiency was sufficiently high (12
). Upon co-transfection with the Cre-expression vector, the lox
P and lox
PV sequences on a transfected vector each recombines specifically with the same lox
sequence on the CA locus, eventually resulting in the replacement of the latter by that derived from the vector in virtually 100% of the cells after both positive and negative selections (14
Here, we describe the development of the ROSA-TET system, which integrates the inducible expression of the Tet-off system, the ubiquitous expression from the ROSA26 locus, and the convenience of the transgene introduction by the RMCE system. The ROSA-TET system is thus an easy-to-use method that enables the establishment of multiple ES cell lines carrying inducible transgenes.